Journal of Virological Methods 202 (2014) 24–27
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Molecular detection of varicella zoster virus while keeping an eye on the budget Khalifa Binkhamis a,b , Turkiya Al-Siyabi a,b , Charles Heinstein a , Todd F. Hatchette a,b , Jason J. LeBlanc a,b,∗ a b
Department of Pathology and Laboratory Medicine, Capital District Health Authority, Halifax, Nova Scotia, Canada Dalhousie University, Halifax, Nova Scotia, Canada
a b s t r a c t Article history: Received 26 October 2013 Received in revised form 7 February 2014 Accepted 11 February 2014 Available online 7 March 2014 Keywords: Homogenization Nucleic acid extraction Real-time PCR Varicella zoster virus Cost analysis
Varicella zoster virus (VZV) PCR is highly sensitive compared to traditional detection methods like culture and direct fluorescent antibody testing (DFA); however, the high cost of commercial assays prohibits their use in many clinical laboratories. Major contributors to cost are the nucleic acid extraction and the PCR reagents. This study evaluated an “in-house” qualitative real-time PCR where the nucleic acid extraction was replaced by a crude extraction, homogenization and heat treatment. Three methods were compared: virus culture and DFA and real-time PCR following each extraction methods. The real-time PCR was highly specific for VZV, and the analytical sensitivity was equivalent following both extraction methods. In contrast, virus culture and DFA was approximately 10,000-fold less sensitive. Using 200 clinical specimens, the sensitivity for the real-time PCR following nucleic acid extraction or homogenization and heat treatment was essentially equivalent at 100% and 97.2%, respectively; whereas, virus culture and DFA was significantly less sensitive at 54.8%. Overall, homogenization and heat treatment combined with a qualitative in-house real-time PCR is a rapid, accurate and cost effective method for the detection of VZV. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Human herpesvirus 3, also known as varicella zoster virus (VZV), is predominately a childhood infection that manifests as a vesicular rash known as chickenpox (Hambleton, 2005). After primary infection the virus latently infects the dorsal root ganglia. With waning immunity or immunosuppression, the virus can reactivate resulting in painful dermatomal lesions known as herpes zoster or shingles (Strommen et al., 1988; Weaver, 2009). Rarely, more severe complications can arise including encephalitis, pneumonia, and disseminated disease in immunocompromized individuals (Weaver, 2009). Laboratory diagnosis of VZV relies on viral culture, DFA, or molecular methods such as PCR (Coffin and Hodinka, 1995; Wilson et al., 2012). With the advent of real-time PCR, rapid results can be achieved with performances that far surpass those of other
methods (Wilson et al., 2012). However, the high cost of commercial PCR and nucleic acid extraction may prohibit routine use in some clinical laboratories. Two strategies have been used to reduce the cost of molecular assays. First, “in-house” molecular assays use relatively inexpensive reagents compared to commercial assays (Espy et al., 2000; Sankuntaw et al., 2011). Secondly, a crude lysis using mechanical disruption with silica beads (i.e. homogenization) and heat treatment was shown to be a cost effective alternative to nucleic acid extraction for the detection of viral DNA from swabs submitted in universal transport media (UTM) (LeBlanc et al., 2012; Al-Siyabi et al., 2013). This study evaluated whether the combination of homogenization with heat treatment and an in-house real-time PCR would be a cost effective strategy for the detection of VZV from viral swabs transported in UTM. 2. Materials and methods
∗ Corresponding author at: Department of Microbiology and Immunology, Room 404C, MacKenzie Building, Queen Elizabeth II (QEII) Health Sciences Centre, 5788 University Avenue, Halifax, Nova Scotia B3H 1V8, Canada. Tel.: +1 902 473 7698; fax: +1 902 473 7971. E-mail addresses: [email protected], [email protected] (J.J. LeBlanc). http://dx.doi.org/10.1016/j.jviromet.2014.02.009 0166-0934/© 2014 Elsevier B.V. All rights reserved.
2.1. Virus culture and DFA Viral cultures and DFA were performed as part of routine testing in the microbiology laboratory at Capital District Health Authority (Halifax, NS, Canada). Briefly, human foreskin fibroblasts
K. Binkhamis et al. / Journal of Virological Methods 202 (2014) 24–27
cells (ATCC CRL-2094) were inoculated with 500 l of specimen and incubated at 37 ◦ C in a 5% CO2 atmosphere. Cells monitored daily and stained by DFA using a Merifluor VZV kit (Meridian BioScience Inc., Villa Cortese, Italy) on day 7 and 14. Fibroblasts were propagated in Minimum Essential Medium containing Earle’s salts, non-essential amino acids and 0.2 mg/ml l-glutamine (Sigma–Aldrich Canada Ltd., Oakville, ON), 2 g/ml amphotericin B (Sigma–Aldrich), 1 mg/ml vancomycin (Sigma–Aldrich), 25 g/ml ampicillin (Novapharm Ltd., Toronto, ON), and 10% fetal calf serum (Hyclone, Thermo Fisher Scientific, Ottawa, ON). 2.2. Homogenization and heat treatment Homogenization and heat treatment was performed as previously described (LeBlanc et al., 2012). Briefly, 500 l of specimen and 0.5 g of acid-washed silica beads (Sigma–Aldrich) were placed on a Fastprep-24 homogenizer (MP BioMedicals, Solon, OH) at 6.5 m/s for 45 s. Following a brief centrifugation at 14,000 × g for 1 min, 200 l of the supernatant was diluted in two volumes of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The homogenate was then heated at 95 ◦ C for 15 min, cooled to room temperature, and 5 l was subjected to VZV real-time PCR. 2.3. Nucleic acid extraction Nucleic acid extractions were performed using manufacturers’ instructions on a MagNAPure LC instrument using 200 l of specimen and a MagNA Pure Total Nucleic Acid Isolation kit (Roche Diagnostics, Mannheim, Germany). Plasmid DNA used for the internal control was purified as described by Al-Siyabi et al. (2013). Nucleic acids were used immediately following extraction and aliquots were placed at −80 ◦ C for long-term storage. 2.4. Qualitative in-house real-time PCR Real-time PCR was performed as duplex reactions with primers and probes targeting the VZV and an internal control termed pGFP (Table S1). Primers were synthesized by Sigma Genosys (Oakville, ON) and probes were purchased from TIB MOLBIOL LLC (Adelphia, NJ). PCR reaction were performed using the LightCycler DNA Master HybProbe kit (Roche Diagnostics) in 20 l reactions consisting of: 5 l of template, 1× LightCycler FastStart mix, 3 mM MgCl2 ; 0.5 U of heat-labile uracil-N-glycosylase (LeBlanc et al., 2008); 5 l pGFP at 400 copies/l; 500 nM of each primer and 300 nM of each probe (Table S1). Amplification and detection were performed using the LightCycler 2.0 instrument under the thermocycling conditions described for the Roche HSV-1/2 detection kit: initial activation at 95 ◦ C for 10 min, followed by 45 amplification cycles of denaturation at 95 ◦ C for 10 s, annealing at 55 ◦ C for 15 s, and elongation at 72 ◦ C for 15 s. Melting temperature (Tm) analysis was performed by measuring the fluorescent signal during the cycling profile: 95 ◦ C for 0 s, 40 ◦ C for 60 s, and 80 ◦ C for 0 s with a 0.2 ◦ C/s transition. Crossing point (Cp) and Tm values were calculated by the manufacturer software. The 640 and 705 nm channels were analyzed for presence or absence of VZV or internal control, respectively. PCR inhibition would be suspected by loss of positivity in the 705 nm channel or a shift in Cp values greater than two standard deviations (approximately ±1.0 Cp) from the value obtained with the negative control.
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extraction method was determined using 10-fold serial dilutions in UTM of a cultured VZV strain Ellen (ATCC VR-1367). Each dilution was simultaneously processed by both extraction methods, and an aliquot immediately inoculated onto cells for virus culture. The limit of detection (LoD) was defined by Probit analysis (Finney, 1971) using triplicate values obtained in three independent experiments (n = 9). Each virus dilution was expressed as 50% tissue culture infective dose (TCID50 )/ml or log10 copies/reaction. Intraand inter-assay reproducibility were calculated and expressed as a percentage of the coefficient of variation (%CV). For quantitation of the viral stock used to determine the analytical sensitivity, the virus dilutions were inoculated onto 96-well plates and after 14 days, stained with DFA to determine the TCID50 /ml. Results represent eight replicates obtained in four independent experiments (n = 32). The virus dilutions were also subjected to quantitative real-time PCR (described below). 2.6. Clinical performance For the clinical evaluation, dermal swabs (n = 200) collected from patients suspected of having VZV infections, between April 2011 and March 2012. The swabs were transported in 3 ml of UTM (Copan Diagnostics Inc., Murrieta, CA) and kept at 4 ◦ C for no more than 24 h prior to processing. Following virus culture, aliquots of the specimens were transferred into anonymized cryotubes and archived at −80 ◦ C for retrospective molecular analyses. To assess sensitivity, specificity, accuracy and precision, the clinical performance of each method was compared to a modified gold standard defined by concordant results (positive or negative) between at least two of the three molecular assays. Specimens with discordant results during method comparison were subjected to discrepant analysis using quantitative real-time PCR using an Artus LC VZV PCR kit (Qiagen Inc., Hilden, Germany) following DNA extraction with a QIAamp DNA Blood Mini kit (Qiagen, Toronto, ON), as recommended by the manufacturer. 2.7. Cost analysis At our institution, the average number of specimens submitted yearly for VZV testing is 676 (range 416–988 for years 2009 to 2012). The average turnaround time for virus culture is 15.6 days (ranging from 7 to 19). A cost analysis was performed that assumed a more practical approach of bi-weekly molecular testing of three to five specimens per run, and three controls (positive, negative and reagent controls). 2.8. Statistical analysis Chi-square and two-tailed Fisher’s exact tests were used to compare proportions in 2-by-2 contingency tables. Binomial confidence intervals (95%) for the clinical parameters are computed by a general method based on “constant chi-square boundaries” (Fleiss et al., 2003). Agreement between assays was measured using kappa statistics. The Statistical Package for Social Sciences (SPSS) software v.10 was used and P ≤ 0.05 was used to denote a statistically significance. 3. Results 3.1. Analytical performance characteristics
2.5. Analytical methods The analytical specificity was determined using high titer nucleic acids that were extracted from human herpes virus types 1–8, or various other microorganisms (Table S2). The analytical sensitivity of virus culture and DFA, or real-time PCR following either
Regardless of the extraction protocol, the in-house realtime PCR was highly specific, with no cross reactions observed with other human herpes viruses or microorganisms (Table S2). The PCR readily detected VZV strain Ellen and vaccine strain OKA. At a probability of 95%, the LoD for virus culture and
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with low Cp values (range 20–27) and virus culture-negative/PCRpositive specimens had Cp values that fell below the limit of detection for virus culture and DFA (Cp ≥ 27). All PCR-positive results had Tm values consistent with the average ± S.D. for the VZV target (68.44 ± 0.48 ◦ C and 68.82 ± 0.57 ◦ C for the extractionand homogenization-based assays, respectively). No PCR inhibition was observed in this study. 3.3. Cost analysis
Fig. 1. Analytical sensitivity determined by Probit analysis. At a probability of 95%, the estimated limit of detection for the homogenization- and NAE-based protocols were 389 copies/ml (log10 = 2.59) and 372 copies/ml (log10 = 2.57), respectively. The analytical sensitivity for virus culture and DFA was approximately 4.4 × 106 copies/ml (log10 = 6.64). Abbreviations: direct fluorescent antibody (DFA); homogenization and heat treatment (HH); nucleic acid extraction (NAE); real-time PCR (rtPCR); and virus culture (VC).
DFA was estimated at 4.4 × 106 copies/ml (log10 = 6.64) or 1.0 TCID50 /ml. The LoD for the real-time PCR following nucleic acid extraction [372 copies/ml (log10 = 2.57) or 0.01 TCID50 /ml] or following homogenization and heat treatment [389 copies/ml (log10 = 2.59) or 0.01 TCID50 /ml] were equivalent (Fig. 1). Positive PCR reactions were also frequently observed using virus dilutions of 0.001 TCID50 /ml (3/9 and 2/94, respectively). By plotting the Cp values over the concentration, overlapping linear relationships were observed (y = −1.9724x + 32.617; R2 = 0.9984 and y = −1.7196x + 31.749; R2 = 0.9967, respectively) that spanned six orders of magnitude (102 to 108 copies/ml) with Cp values ranging from 20 to 31 (data not shown). The intra- and inter-assay reproducibility of the real-time PCR following homogenization and heat treatment or nucleic acid extraction ranged from 0.02 to 2.57%, and 0.03 to 3.01%, respectively. Similarly, intra- and inter-assay reproducibility ranged from 0.01 to 2.17% and 1.1 to 2.47%. 3.2. Clinical performance For the clinical validation, swabs in UTM submitted for VZV detection were processed by virus culture and DFA, and aliquots were stored at −80 ◦ C for retrospective molecular analyses. Forty virus culture-positive specimens and 160 virus culture-negative specimens were randomly selected and evaluated using real-time PCR following both extraction methods. When comparing virus culture to PCR following either extraction methods, there were 127 concordant negative and 40 concordant positive results. Thirty-one additional positives were detected by PCR following homogenization and heat treatment and another two using nucleic acid extraction. All 33 discordant results had Tm values consistent with the VZV target and the results were confirmed by manual DNA extraction and commercial quantitative PCR. Compared to the modified gold standard, the sensitivity of the real-time PCR following homogenization and heat treatment or nucleic acid extraction was equivalent at 97.2% and 100%, respectively (Table 1). Virus culture and DFA was significantly (P ≤ 0.001) less sensitive at 54.8% (Table 1). While the PCR assays had high accuracy (99% and 100%), the low accuracy of virus culture and DFA (83.5%) can be explained by its poor sensitivity (Table). As expected, virus culture-positive specimens had positive real-time PCR results
The average cost of a commercial PCR following nucleic acid extraction would range from $45 to $55 per specimen. In comparison, the in-house real-time PCR following a nucleic acid extraction would reduce the cost approximately 2-fold ($18.05 to $25.97). Replacement of nucleic acid extraction with homogenization and heat treatment further reduces the cost by approximately 50% ($7.93 to $9.63), which is comparable to the average cost of virus culture ($9.27 to 10.46). While labor was not included in the analysis, the “hands on” time required for bi-weekly processing for either molecular method is far less than the time required for maintenance and processing of virus culture and DFA. 4. Discussion Compared to virus culture, real-time PCRs offer rapid results with high sensitivity and specificity, making them ideal diagnostic tests. Despite these advantages, molecular testing is relatively expensive, with both the extraction and PCR reagents contributing to the cost. Wilson et al. (2012) recently reported high sensitivity and specificity for PCR compared to virus culture and DFA, yet the high cost of PCR prompted a recommendation for a testing algorithm based primarily on DFA. This study provides an option for the primary detection of VZV using PCR, at a cost comparable to virus culture and DFA using a crude extraction and an inexpensive in-house qualitative real-time PCR assay. The analytical performance characteristics of the real-time PCR following homogenization and heat treatment or nucleic acid extraction were equivalent and far exceeded those obtained with virus culture and DFA (Fig. 1 and Table 1). Both molecular methods demonstrated excellent analytical specificity (Table S2) and their analytical sensitivity was approximately 10,000-fold more sensitive than virus culture and DFA (Fig. 1). The high sensitivity of the real-time PCR, as well as the low intra- and inter-assay variations are consistent with values previously reported (Sankuntaw et al., 2011). The clinical sensitivity of the PCR following homogenization and heat treatment and nucleic acid extraction was 97.2% and 100%, respectively (Table 1), which far surpasses the performance of virus culture and DFA with a dismal sensitivity of 54.8%. The 45% increase in sensitivity when transitioning from virus culture to molecular testing is not surprising since similar results were obtained by other laboratories for VZV and other viruses (Wilson et al., 2012; LeBlanc et al., 2012; Al-Siyabi et al., 2013; Campbell et al., 2008). Unlike the low clinical sensitivity observed with virus culture and DFA, the 2.8% difference between the molecular assays was not significant. The two additional positive results obtained using nucleic acid extraction had Cp values near the LoD (32.56 and 35.60), suggesting the slight difference might be attributed to a Poisson distribution with low viral loads (Sykes et al., 1992). The two false negative results obtained with homogenization were not attributed to PCR inhibition. No PCR inhibition was observed in this study. However, rates of 0.5 to 0.6% have been reported with both extraction methods suggesting that PCR inhibition should be monitored (LeBlanc et al., 2012; Al-Siyabi et al., 2013). With the clinical sensitivities described above, and assuming a yearly average of 676 specimens with a positivity rate for virus
K. Binkhamis et al. / Journal of Virological Methods 202 (2014) 24–27
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Table 1 Summary of the method performance characteristics compared to the modified gold standard.a Method Virus culture and DFA Homogenization and heat treatment and real-time PCR Nucleic acid extraction and real-time PCR
Sensitivity % (95% CI)
Accuracy % (95% CI)
Precision % (95% CI)
Kappa (95% CI)
54.8 (49.3–54.8) 97.3 (92.9–97.3)
Specificity % (95% CI) 100.0 (96.8–100.0) 100.0 (97.5–100.0)
83.5 (79.5–83.5) 99.0 (95.8–99.0)
100 (90.0–100.0) 100.0 (95.5–100.0)
0.606 (0.511–0.606) 0.978 (0.909–0.978)
100.0 (97.2–100.0)
100.0 (97.8–100.0)
100.0 (97.2–100.0)
100.0 (96.2–100.0)
1.000 (0.940–1.000)
a A case was defined by concordant results (positive or negative) between at least two assays. A manual nucleic acid extraction and commercial real-time PCR was used to resolve discrepant results. Abbreviations: confidence interval (CI); homogenization and heat treatment (HH); nucleic acid extraction (NAE); and real-time PCR (rtPCR).
culture and DFA of 18.2%, the real-time PCR following nucleic acid extraction or homogenization and heat treatment should yield 101 and 95 additional positives yearly. Taking fiscal responsibilities into consideration, the incremental cost expended to detect the six additional positives using the nucleic acid extraction (presumed VZV-negative by the homogenization assay) would be $2124.30, a value that is unacceptably high for a questionable benefit. With a bi-weekly testing schedule, the average specimen turnaround times can be reduced from 16 days to 3–4 days. Since identical thermocycling conditions were used for VZV, HSV, and adenovirus, further workflow efficiencies can be gained through concurrent testing of multiple viruses on the same instrument (LeBlanc et al., 2012; Al-Siyabi et al., 2013). Future experiments will examine whether homogenization can be applied to other specimen types and whether the real-time PCR can be multiplexed to include other herpes viruses (Sankuntaw et al., 2011; Stöcher et al., 2004). Overall, the data from this study suggests that virus culture and DFA should be discontinued and VZV detection performed with sensitive molecular assays. Unlike the high cost of commercial PCR assays, this study demonstrated a cost effective method for the molecular detection VZV by coupling an in-house qualitative realtime PCR to a low cost extraction using homogenization and heat treatment. Conflict of interest None of the authors have any conflict of interest related to the content of this manuscript and all authors agree to its content. Funding This project was supported by the Division of Microbiology, Department of Pathology and Laboratory Medicine, Capital District Health Authority (Halifax, NS, Canada) as part of quality improvement for the laboratory detection of VZV. Ethical approval Since the purpose of this clinical validation was quality improvement of the laboratory detection of VZV and relied exclusively on anonymous human biological materials that did not use or generate identifiable patient information, research ethics board (REB) review was not required based on Chapter 2, article 2.4 of the TriCouncil Policy Statement: Ethical Conduct for Research Involving Humans (2nd edition). Acknowledgements We would like to thank members of Division of Microbiology, Department of Pathology and Laboratory Medicine at Capital District Health Authority (Halifax, Nova Scotia) for their ongoing support. In particular, we are indebted to Wanda Brewer for the propagation and maintenance of HFF cells, and the various
technologists responsible for routine virus culture. We would like to thank Dr. Wenda Greer (Division of Hematopathology, Department of Pathology and Laboratory Medicine at Capital District Health Authority (Halifax, Nova Scotia) for the human herpes virus 4, Dr. Craig MacCormick (Dalhousie University, Halifax, NS) for human herpes virus 8, Dr. Raymond Tellier and Salleen Wong from the Provincial Laboratory for Public Health (Calgary, Alberta) for human herpes viruses 6a, 6b, and 7. We are also indebted to Nathalie Bastien at the National Microbiology Laboratory for the vaccine strain of varicella zoster virus (strain OKA). None of the authors have any financial conflicts of interest to declare and agree with the final content of this manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.jviromet.2014.02.009. References Al-Siyabi, T., Binkhamis, K., Wilcox, M., Wong, S., Pabbaraju, K., Tellier, R., Hatchette, T.F., LeBlanc, J.J., 2013. A cost effective real-time PCR for the detection of adenovirus from viral swabs. Virol. J. 10, 184–196. Campbell, S., Leblanc, J., Pettipas, J., Hatchette, T., Davidson, R., 2008. A comparison of cell culture versus real-time PCR for the detection of HSV1/2 from routine clinical specimens. Can. J. Infect. Dis. Med. Microbiol. 19, 77–142. Coffin, S.E., Hodinka, R.L., 1995. Utility of direct immunofluorescence and virus culture for detection of varicella-zoster virus in skin lesions. J. Clin. Microbiol. 33, 2792–2795. Espy, M.J., Teo, R., Ross, T.K., Svien, K.A., Wold, A.D., Uhl, J.R., Smith, T.F., 2000. Diagnosis of varicella-zoster virus infections in the clinical laboratory by LightCycler PCR. J. Clin. Microbiol. 38, 3187–3189. Finney, D.J., 1971. Probit Analysis, 3rd ed. Cambridge University Press, Cambridge, UK. Fleiss, J.L., Levin, B., Paik, M.C., 2003. In: Shewart, W.A., Wilks, S.S. (Eds.), Statistical Methods for Rates and Proportions. John Wiley & Sons, New York. Hambleton, S., 2005. Chickenpox. Curr. Opin. Infect. Dis. 18, 235–240. LeBlanc, J., Pettipas, J., Campbell, S., Davidson, R.J., Hatchette, T.F., 2008. Uracil-DNA glycosylase (UNG) influences the melting curve profiles of herpes simplex virus (HSV) hybridization probes. J. Virol. Methods 151, 158–160. LeBlanc, J.J., Heinstein, C.R., Hatchette, T.F., 2012. Homogenization with heat treatment: a cost effective alternative to nucleic acid extraction for herpes simplex virus real-time PCR from viral swabs. J. Virol. Methods 179, 261–264. Sankuntaw, N., Sukprasert, S., Engchanil, C., Kaewkes, W., Chantratita, W., Pairoj, V., Lulitanond, V., 2011. Single-tube multiplex real-time PCR for the rapid detection of herpesvirus infections of the central nervous system. Mol. Cell. Probes 25, 114–120. Strommen, G.L., Pucino, F., Tight, R.R., Beck, C.L., 1988. Human infection with herpes zoster: etiology, pathophysiology, diagnosis, clinical course, and treatment. Pharmacotherapy 8, 52–68. Stöcher, M., Hölzl, G., Stekel, H., Berg, J., 2004. Automated detection of five human herpes virus DNAs by a set of LightCycler PCRs complemented with a single multiple internal control. J. Clin. Virol. 29, 171–178. Sykes, P.J., Neoh, S.H., Brisco, M.J., Hughes, E., Condon, J., Morley, A.A., 1992. Quantitation of targets for PCR by use of limiting dilution. BioTechniques 13, 444–449. Weaver, B.A., 2009. Herpes zoster overview: natural history and incidence. J. Am. Osteopath. Assoc. 109, S2–S6. Wilson, D.A., Yen-Lieberman, B., Schindler, S., Asamoto, K., Schold, J.D., Procop, G.W., 2012. Should varicella-zoster virus culture be eliminated? A comparison of direct immunofluorescence antigen detection, culture, and PCR, with a historical review. J. Clin. Microbiol. 50, 4120–4122.
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Molecular detection of varicella zoster virus while keeping an eye on the budget Khalifa Binkhamis a,b , Turkiya Al-Siyabi a,b , Charles Heinstein a , Todd F. Hatchette a,b , Jason J. LeBlanc a,b,∗ a b
Department of Pathology and Laboratory Medicine, Capital District Health Authority, Halifax, Nova Scotia, Canada Dalhousie University, Halifax, Nova Scotia, Canada
a b s t r a c t Article history: Received 26 October 2013 Received in revised form 7 February 2014 Accepted 11 February 2014 Available online 7 March 2014 Keywords: Homogenization Nucleic acid extraction Real-time PCR Varicella zoster virus Cost analysis
Varicella zoster virus (VZV) PCR is highly sensitive compared to traditional detection methods like culture and direct fluorescent antibody testing (DFA); however, the high cost of commercial assays prohibits their use in many clinical laboratories. Major contributors to cost are the nucleic acid extraction and the PCR reagents. This study evaluated an “in-house” qualitative real-time PCR where the nucleic acid extraction was replaced by a crude extraction, homogenization and heat treatment. Three methods were compared: virus culture and DFA and real-time PCR following each extraction methods. The real-time PCR was highly specific for VZV, and the analytical sensitivity was equivalent following both extraction methods. In contrast, virus culture and DFA was approximately 10,000-fold less sensitive. Using 200 clinical specimens, the sensitivity for the real-time PCR following nucleic acid extraction or homogenization and heat treatment was essentially equivalent at 100% and 97.2%, respectively; whereas, virus culture and DFA was significantly less sensitive at 54.8%. Overall, homogenization and heat treatment combined with a qualitative in-house real-time PCR is a rapid, accurate and cost effective method for the detection of VZV. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Human herpesvirus 3, also known as varicella zoster virus (VZV), is predominately a childhood infection that manifests as a vesicular rash known as chickenpox (Hambleton, 2005). After primary infection the virus latently infects the dorsal root ganglia. With waning immunity or immunosuppression, the virus can reactivate resulting in painful dermatomal lesions known as herpes zoster or shingles (Strommen et al., 1988; Weaver, 2009). Rarely, more severe complications can arise including encephalitis, pneumonia, and disseminated disease in immunocompromized individuals (Weaver, 2009). Laboratory diagnosis of VZV relies on viral culture, DFA, or molecular methods such as PCR (Coffin and Hodinka, 1995; Wilson et al., 2012). With the advent of real-time PCR, rapid results can be achieved with performances that far surpass those of other
methods (Wilson et al., 2012). However, the high cost of commercial PCR and nucleic acid extraction may prohibit routine use in some clinical laboratories. Two strategies have been used to reduce the cost of molecular assays. First, “in-house” molecular assays use relatively inexpensive reagents compared to commercial assays (Espy et al., 2000; Sankuntaw et al., 2011). Secondly, a crude lysis using mechanical disruption with silica beads (i.e. homogenization) and heat treatment was shown to be a cost effective alternative to nucleic acid extraction for the detection of viral DNA from swabs submitted in universal transport media (UTM) (LeBlanc et al., 2012; Al-Siyabi et al., 2013). This study evaluated whether the combination of homogenization with heat treatment and an in-house real-time PCR would be a cost effective strategy for the detection of VZV from viral swabs transported in UTM. 2. Materials and methods
∗ Corresponding author at: Department of Microbiology and Immunology, Room 404C, MacKenzie Building, Queen Elizabeth II (QEII) Health Sciences Centre, 5788 University Avenue, Halifax, Nova Scotia B3H 1V8, Canada. Tel.: +1 902 473 7698; fax: +1 902 473 7971. E-mail addresses: [email protected], [email protected] (J.J. LeBlanc). http://dx.doi.org/10.1016/j.jviromet.2014.02.009 0166-0934/© 2014 Elsevier B.V. All rights reserved.
2.1. Virus culture and DFA Viral cultures and DFA were performed as part of routine testing in the microbiology laboratory at Capital District Health Authority (Halifax, NS, Canada). Briefly, human foreskin fibroblasts
K. Binkhamis et al. / Journal of Virological Methods 202 (2014) 24–27
cells (ATCC CRL-2094) were inoculated with 500 l of specimen and incubated at 37 ◦ C in a 5% CO2 atmosphere. Cells monitored daily and stained by DFA using a Merifluor VZV kit (Meridian BioScience Inc., Villa Cortese, Italy) on day 7 and 14. Fibroblasts were propagated in Minimum Essential Medium containing Earle’s salts, non-essential amino acids and 0.2 mg/ml l-glutamine (Sigma–Aldrich Canada Ltd., Oakville, ON), 2 g/ml amphotericin B (Sigma–Aldrich), 1 mg/ml vancomycin (Sigma–Aldrich), 25 g/ml ampicillin (Novapharm Ltd., Toronto, ON), and 10% fetal calf serum (Hyclone, Thermo Fisher Scientific, Ottawa, ON). 2.2. Homogenization and heat treatment Homogenization and heat treatment was performed as previously described (LeBlanc et al., 2012). Briefly, 500 l of specimen and 0.5 g of acid-washed silica beads (Sigma–Aldrich) were placed on a Fastprep-24 homogenizer (MP BioMedicals, Solon, OH) at 6.5 m/s for 45 s. Following a brief centrifugation at 14,000 × g for 1 min, 200 l of the supernatant was diluted in two volumes of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The homogenate was then heated at 95 ◦ C for 15 min, cooled to room temperature, and 5 l was subjected to VZV real-time PCR. 2.3. Nucleic acid extraction Nucleic acid extractions were performed using manufacturers’ instructions on a MagNAPure LC instrument using 200 l of specimen and a MagNA Pure Total Nucleic Acid Isolation kit (Roche Diagnostics, Mannheim, Germany). Plasmid DNA used for the internal control was purified as described by Al-Siyabi et al. (2013). Nucleic acids were used immediately following extraction and aliquots were placed at −80 ◦ C for long-term storage. 2.4. Qualitative in-house real-time PCR Real-time PCR was performed as duplex reactions with primers and probes targeting the VZV and an internal control termed pGFP (Table S1). Primers were synthesized by Sigma Genosys (Oakville, ON) and probes were purchased from TIB MOLBIOL LLC (Adelphia, NJ). PCR reaction were performed using the LightCycler DNA Master HybProbe kit (Roche Diagnostics) in 20 l reactions consisting of: 5 l of template, 1× LightCycler FastStart mix, 3 mM MgCl2 ; 0.5 U of heat-labile uracil-N-glycosylase (LeBlanc et al., 2008); 5 l pGFP at 400 copies/l; 500 nM of each primer and 300 nM of each probe (Table S1). Amplification and detection were performed using the LightCycler 2.0 instrument under the thermocycling conditions described for the Roche HSV-1/2 detection kit: initial activation at 95 ◦ C for 10 min, followed by 45 amplification cycles of denaturation at 95 ◦ C for 10 s, annealing at 55 ◦ C for 15 s, and elongation at 72 ◦ C for 15 s. Melting temperature (Tm) analysis was performed by measuring the fluorescent signal during the cycling profile: 95 ◦ C for 0 s, 40 ◦ C for 60 s, and 80 ◦ C for 0 s with a 0.2 ◦ C/s transition. Crossing point (Cp) and Tm values were calculated by the manufacturer software. The 640 and 705 nm channels were analyzed for presence or absence of VZV or internal control, respectively. PCR inhibition would be suspected by loss of positivity in the 705 nm channel or a shift in Cp values greater than two standard deviations (approximately ±1.0 Cp) from the value obtained with the negative control.
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extraction method was determined using 10-fold serial dilutions in UTM of a cultured VZV strain Ellen (ATCC VR-1367). Each dilution was simultaneously processed by both extraction methods, and an aliquot immediately inoculated onto cells for virus culture. The limit of detection (LoD) was defined by Probit analysis (Finney, 1971) using triplicate values obtained in three independent experiments (n = 9). Each virus dilution was expressed as 50% tissue culture infective dose (TCID50 )/ml or log10 copies/reaction. Intraand inter-assay reproducibility were calculated and expressed as a percentage of the coefficient of variation (%CV). For quantitation of the viral stock used to determine the analytical sensitivity, the virus dilutions were inoculated onto 96-well plates and after 14 days, stained with DFA to determine the TCID50 /ml. Results represent eight replicates obtained in four independent experiments (n = 32). The virus dilutions were also subjected to quantitative real-time PCR (described below). 2.6. Clinical performance For the clinical evaluation, dermal swabs (n = 200) collected from patients suspected of having VZV infections, between April 2011 and March 2012. The swabs were transported in 3 ml of UTM (Copan Diagnostics Inc., Murrieta, CA) and kept at 4 ◦ C for no more than 24 h prior to processing. Following virus culture, aliquots of the specimens were transferred into anonymized cryotubes and archived at −80 ◦ C for retrospective molecular analyses. To assess sensitivity, specificity, accuracy and precision, the clinical performance of each method was compared to a modified gold standard defined by concordant results (positive or negative) between at least two of the three molecular assays. Specimens with discordant results during method comparison were subjected to discrepant analysis using quantitative real-time PCR using an Artus LC VZV PCR kit (Qiagen Inc., Hilden, Germany) following DNA extraction with a QIAamp DNA Blood Mini kit (Qiagen, Toronto, ON), as recommended by the manufacturer. 2.7. Cost analysis At our institution, the average number of specimens submitted yearly for VZV testing is 676 (range 416–988 for years 2009 to 2012). The average turnaround time for virus culture is 15.6 days (ranging from 7 to 19). A cost analysis was performed that assumed a more practical approach of bi-weekly molecular testing of three to five specimens per run, and three controls (positive, negative and reagent controls). 2.8. Statistical analysis Chi-square and two-tailed Fisher’s exact tests were used to compare proportions in 2-by-2 contingency tables. Binomial confidence intervals (95%) for the clinical parameters are computed by a general method based on “constant chi-square boundaries” (Fleiss et al., 2003). Agreement between assays was measured using kappa statistics. The Statistical Package for Social Sciences (SPSS) software v.10 was used and P ≤ 0.05 was used to denote a statistically significance. 3. Results 3.1. Analytical performance characteristics
2.5. Analytical methods The analytical specificity was determined using high titer nucleic acids that were extracted from human herpes virus types 1–8, or various other microorganisms (Table S2). The analytical sensitivity of virus culture and DFA, or real-time PCR following either
Regardless of the extraction protocol, the in-house realtime PCR was highly specific, with no cross reactions observed with other human herpes viruses or microorganisms (Table S2). The PCR readily detected VZV strain Ellen and vaccine strain OKA. At a probability of 95%, the LoD for virus culture and
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with low Cp values (range 20–27) and virus culture-negative/PCRpositive specimens had Cp values that fell below the limit of detection for virus culture and DFA (Cp ≥ 27). All PCR-positive results had Tm values consistent with the average ± S.D. for the VZV target (68.44 ± 0.48 ◦ C and 68.82 ± 0.57 ◦ C for the extractionand homogenization-based assays, respectively). No PCR inhibition was observed in this study. 3.3. Cost analysis
Fig. 1. Analytical sensitivity determined by Probit analysis. At a probability of 95%, the estimated limit of detection for the homogenization- and NAE-based protocols were 389 copies/ml (log10 = 2.59) and 372 copies/ml (log10 = 2.57), respectively. The analytical sensitivity for virus culture and DFA was approximately 4.4 × 106 copies/ml (log10 = 6.64). Abbreviations: direct fluorescent antibody (DFA); homogenization and heat treatment (HH); nucleic acid extraction (NAE); real-time PCR (rtPCR); and virus culture (VC).
DFA was estimated at 4.4 × 106 copies/ml (log10 = 6.64) or 1.0 TCID50 /ml. The LoD for the real-time PCR following nucleic acid extraction [372 copies/ml (log10 = 2.57) or 0.01 TCID50 /ml] or following homogenization and heat treatment [389 copies/ml (log10 = 2.59) or 0.01 TCID50 /ml] were equivalent (Fig. 1). Positive PCR reactions were also frequently observed using virus dilutions of 0.001 TCID50 /ml (3/9 and 2/94, respectively). By plotting the Cp values over the concentration, overlapping linear relationships were observed (y = −1.9724x + 32.617; R2 = 0.9984 and y = −1.7196x + 31.749; R2 = 0.9967, respectively) that spanned six orders of magnitude (102 to 108 copies/ml) with Cp values ranging from 20 to 31 (data not shown). The intra- and inter-assay reproducibility of the real-time PCR following homogenization and heat treatment or nucleic acid extraction ranged from 0.02 to 2.57%, and 0.03 to 3.01%, respectively. Similarly, intra- and inter-assay reproducibility ranged from 0.01 to 2.17% and 1.1 to 2.47%. 3.2. Clinical performance For the clinical validation, swabs in UTM submitted for VZV detection were processed by virus culture and DFA, and aliquots were stored at −80 ◦ C for retrospective molecular analyses. Forty virus culture-positive specimens and 160 virus culture-negative specimens were randomly selected and evaluated using real-time PCR following both extraction methods. When comparing virus culture to PCR following either extraction methods, there were 127 concordant negative and 40 concordant positive results. Thirty-one additional positives were detected by PCR following homogenization and heat treatment and another two using nucleic acid extraction. All 33 discordant results had Tm values consistent with the VZV target and the results were confirmed by manual DNA extraction and commercial quantitative PCR. Compared to the modified gold standard, the sensitivity of the real-time PCR following homogenization and heat treatment or nucleic acid extraction was equivalent at 97.2% and 100%, respectively (Table 1). Virus culture and DFA was significantly (P ≤ 0.001) less sensitive at 54.8% (Table 1). While the PCR assays had high accuracy (99% and 100%), the low accuracy of virus culture and DFA (83.5%) can be explained by its poor sensitivity (Table). As expected, virus culture-positive specimens had positive real-time PCR results
The average cost of a commercial PCR following nucleic acid extraction would range from $45 to $55 per specimen. In comparison, the in-house real-time PCR following a nucleic acid extraction would reduce the cost approximately 2-fold ($18.05 to $25.97). Replacement of nucleic acid extraction with homogenization and heat treatment further reduces the cost by approximately 50% ($7.93 to $9.63), which is comparable to the average cost of virus culture ($9.27 to 10.46). While labor was not included in the analysis, the “hands on” time required for bi-weekly processing for either molecular method is far less than the time required for maintenance and processing of virus culture and DFA. 4. Discussion Compared to virus culture, real-time PCRs offer rapid results with high sensitivity and specificity, making them ideal diagnostic tests. Despite these advantages, molecular testing is relatively expensive, with both the extraction and PCR reagents contributing to the cost. Wilson et al. (2012) recently reported high sensitivity and specificity for PCR compared to virus culture and DFA, yet the high cost of PCR prompted a recommendation for a testing algorithm based primarily on DFA. This study provides an option for the primary detection of VZV using PCR, at a cost comparable to virus culture and DFA using a crude extraction and an inexpensive in-house qualitative real-time PCR assay. The analytical performance characteristics of the real-time PCR following homogenization and heat treatment or nucleic acid extraction were equivalent and far exceeded those obtained with virus culture and DFA (Fig. 1 and Table 1). Both molecular methods demonstrated excellent analytical specificity (Table S2) and their analytical sensitivity was approximately 10,000-fold more sensitive than virus culture and DFA (Fig. 1). The high sensitivity of the real-time PCR, as well as the low intra- and inter-assay variations are consistent with values previously reported (Sankuntaw et al., 2011). The clinical sensitivity of the PCR following homogenization and heat treatment and nucleic acid extraction was 97.2% and 100%, respectively (Table 1), which far surpasses the performance of virus culture and DFA with a dismal sensitivity of 54.8%. The 45% increase in sensitivity when transitioning from virus culture to molecular testing is not surprising since similar results were obtained by other laboratories for VZV and other viruses (Wilson et al., 2012; LeBlanc et al., 2012; Al-Siyabi et al., 2013; Campbell et al., 2008). Unlike the low clinical sensitivity observed with virus culture and DFA, the 2.8% difference between the molecular assays was not significant. The two additional positive results obtained using nucleic acid extraction had Cp values near the LoD (32.56 and 35.60), suggesting the slight difference might be attributed to a Poisson distribution with low viral loads (Sykes et al., 1992). The two false negative results obtained with homogenization were not attributed to PCR inhibition. No PCR inhibition was observed in this study. However, rates of 0.5 to 0.6% have been reported with both extraction methods suggesting that PCR inhibition should be monitored (LeBlanc et al., 2012; Al-Siyabi et al., 2013). With the clinical sensitivities described above, and assuming a yearly average of 676 specimens with a positivity rate for virus
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Table 1 Summary of the method performance characteristics compared to the modified gold standard.a Method Virus culture and DFA Homogenization and heat treatment and real-time PCR Nucleic acid extraction and real-time PCR
Sensitivity % (95% CI)
Accuracy % (95% CI)
Precision % (95% CI)
Kappa (95% CI)
54.8 (49.3–54.8) 97.3 (92.9–97.3)
Specificity % (95% CI) 100.0 (96.8–100.0) 100.0 (97.5–100.0)
83.5 (79.5–83.5) 99.0 (95.8–99.0)
100 (90.0–100.0) 100.0 (95.5–100.0)
0.606 (0.511–0.606) 0.978 (0.909–0.978)
100.0 (97.2–100.0)
100.0 (97.8–100.0)
100.0 (97.2–100.0)
100.0 (96.2–100.0)
1.000 (0.940–1.000)
a A case was defined by concordant results (positive or negative) between at least two assays. A manual nucleic acid extraction and commercial real-time PCR was used to resolve discrepant results. Abbreviations: confidence interval (CI); homogenization and heat treatment (HH); nucleic acid extraction (NAE); and real-time PCR (rtPCR).
culture and DFA of 18.2%, the real-time PCR following nucleic acid extraction or homogenization and heat treatment should yield 101 and 95 additional positives yearly. Taking fiscal responsibilities into consideration, the incremental cost expended to detect the six additional positives using the nucleic acid extraction (presumed VZV-negative by the homogenization assay) would be $2124.30, a value that is unacceptably high for a questionable benefit. With a bi-weekly testing schedule, the average specimen turnaround times can be reduced from 16 days to 3–4 days. Since identical thermocycling conditions were used for VZV, HSV, and adenovirus, further workflow efficiencies can be gained through concurrent testing of multiple viruses on the same instrument (LeBlanc et al., 2012; Al-Siyabi et al., 2013). Future experiments will examine whether homogenization can be applied to other specimen types and whether the real-time PCR can be multiplexed to include other herpes viruses (Sankuntaw et al., 2011; Stöcher et al., 2004). Overall, the data from this study suggests that virus culture and DFA should be discontinued and VZV detection performed with sensitive molecular assays. Unlike the high cost of commercial PCR assays, this study demonstrated a cost effective method for the molecular detection VZV by coupling an in-house qualitative realtime PCR to a low cost extraction using homogenization and heat treatment. Conflict of interest None of the authors have any conflict of interest related to the content of this manuscript and all authors agree to its content. Funding This project was supported by the Division of Microbiology, Department of Pathology and Laboratory Medicine, Capital District Health Authority (Halifax, NS, Canada) as part of quality improvement for the laboratory detection of VZV. Ethical approval Since the purpose of this clinical validation was quality improvement of the laboratory detection of VZV and relied exclusively on anonymous human biological materials that did not use or generate identifiable patient information, research ethics board (REB) review was not required based on Chapter 2, article 2.4 of the TriCouncil Policy Statement: Ethical Conduct for Research Involving Humans (2nd edition). Acknowledgements We would like to thank members of Division of Microbiology, Department of Pathology and Laboratory Medicine at Capital District Health Authority (Halifax, Nova Scotia) for their ongoing support. In particular, we are indebted to Wanda Brewer for the propagation and maintenance of HFF cells, and the various
technologists responsible for routine virus culture. We would like to thank Dr. Wenda Greer (Division of Hematopathology, Department of Pathology and Laboratory Medicine at Capital District Health Authority (Halifax, Nova Scotia) for the human herpes virus 4, Dr. Craig MacCormick (Dalhousie University, Halifax, NS) for human herpes virus 8, Dr. Raymond Tellier and Salleen Wong from the Provincial Laboratory for Public Health (Calgary, Alberta) for human herpes viruses 6a, 6b, and 7. We are also indebted to Nathalie Bastien at the National Microbiology Laboratory for the vaccine strain of varicella zoster virus (strain OKA). None of the authors have any financial conflicts of interest to declare and agree with the final content of this manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.jviromet.2014.02.009. References Al-Siyabi, T., Binkhamis, K., Wilcox, M., Wong, S., Pabbaraju, K., Tellier, R., Hatchette, T.F., LeBlanc, J.J., 2013. A cost effective real-time PCR for the detection of adenovirus from viral swabs. Virol. J. 10, 184–196. Campbell, S., Leblanc, J., Pettipas, J., Hatchette, T., Davidson, R., 2008. A comparison of cell culture versus real-time PCR for the detection of HSV1/2 from routine clinical specimens. Can. J. Infect. Dis. Med. Microbiol. 19, 77–142. Coffin, S.E., Hodinka, R.L., 1995. Utility of direct immunofluorescence and virus culture for detection of varicella-zoster virus in skin lesions. J. Clin. Microbiol. 33, 2792–2795. Espy, M.J., Teo, R., Ross, T.K., Svien, K.A., Wold, A.D., Uhl, J.R., Smith, T.F., 2000. Diagnosis of varicella-zoster virus infections in the clinical laboratory by LightCycler PCR. J. Clin. Microbiol. 38, 3187–3189. Finney, D.J., 1971. Probit Analysis, 3rd ed. Cambridge University Press, Cambridge, UK. Fleiss, J.L., Levin, B., Paik, M.C., 2003. In: Shewart, W.A., Wilks, S.S. (Eds.), Statistical Methods for Rates and Proportions. John Wiley & Sons, New York. Hambleton, S., 2005. Chickenpox. Curr. Opin. Infect. Dis. 18, 235–240. LeBlanc, J., Pettipas, J., Campbell, S., Davidson, R.J., Hatchette, T.F., 2008. Uracil-DNA glycosylase (UNG) influences the melting curve profiles of herpes simplex virus (HSV) hybridization probes. J. Virol. Methods 151, 158–160. LeBlanc, J.J., Heinstein, C.R., Hatchette, T.F., 2012. Homogenization with heat treatment: a cost effective alternative to nucleic acid extraction for herpes simplex virus real-time PCR from viral swabs. J. Virol. Methods 179, 261–264. Sankuntaw, N., Sukprasert, S., Engchanil, C., Kaewkes, W., Chantratita, W., Pairoj, V., Lulitanond, V., 2011. Single-tube multiplex real-time PCR for the rapid detection of herpesvirus infections of the central nervous system. Mol. Cell. Probes 25, 114–120. Strommen, G.L., Pucino, F., Tight, R.R., Beck, C.L., 1988. Human infection with herpes zoster: etiology, pathophysiology, diagnosis, clinical course, and treatment. Pharmacotherapy 8, 52–68. Stöcher, M., Hölzl, G., Stekel, H., Berg, J., 2004. Automated detection of five human herpes virus DNAs by a set of LightCycler PCRs complemented with a single multiple internal control. J. Clin. Virol. 29, 171–178. Sykes, P.J., Neoh, S.H., Brisco, M.J., Hughes, E., Condon, J., Morley, A.A., 1992. Quantitation of targets for PCR by use of limiting dilution. BioTechniques 13, 444–449. Weaver, B.A., 2009. Herpes zoster overview: natural history and incidence. J. Am. Osteopath. Assoc. 109, S2–S6. Wilson, D.A., Yen-Lieberman, B., Schindler, S., Asamoto, K., Schold, J.D., Procop, G.W., 2012. Should varicella-zoster virus culture be eliminated? A comparison of direct immunofluorescence antigen detection, culture, and PCR, with a historical review. J. Clin. Microbiol. 50, 4120–4122.
